EP2281063B1 - Procédé de prédiction d'une réponse clinique d'un patient souffrant d'un cancer ou présentant un risque de développer un cancer vis-à-vis d'un mode de traitement donné - Google Patents

Procédé de prédiction d'une réponse clinique d'un patient souffrant d'un cancer ou présentant un risque de développer un cancer vis-à-vis d'un mode de traitement donné Download PDF

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EP2281063B1
EP2281063B1 EP09737953.1A EP09737953A EP2281063B1 EP 2281063 B1 EP2281063 B1 EP 2281063B1 EP 09737953 A EP09737953 A EP 09737953A EP 2281063 B1 EP2281063 B1 EP 2281063B1
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spon
expression
cancer
expression level
patient
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EP2281063A1 (fr
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Ralph Markus Wirtz
Wolfgang Michael BRÜCKL
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Siemens Healthcare Diagnostics GmbH Germany
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Siemens Healthcare Diagnostics GmbH Germany
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to methods for prediction of the therapeutic success of cancer, preferably colorectal cancer, therapy.
  • Colorectal cancer represents the second leading cause of cancer related deaths in the European Union (Eucan, Cancer Manufacturer Database 1998).
  • Eucan, Cancer Manufacturer Database 1998 One million people worldwide are diagnosed with this cancer annually, about half of them will succumb, mostly to metastatic disease (Globocan, Cancer Manufacturer Database 1998).
  • Globocan, Cancer Manufacturer Database 1998 Though much is known about the genetic pathways leading to colorectal neoplasia, the exact molecular mechanisms underlying tumor growth, local invasion, angiogenesis, intravasation and finally metastasis remain poorly understood.
  • the relevance of these mechanisms for therapy success or failure have not been resolved and prognostic/predictive markers helping to guide therapy decisions have not yet been identified or validated for clinical routine usage with sufficient level of evidence.
  • CRC patients About 75% percent of patients who are diagnosed with CRC undergo curative treatment. The long term survival of CRC patients depends on the local tumor stage and the potential development of synchronous or metachronous distant metastases.
  • the 5-year-survival rate of CRC patients exceeds 90% in the UlCC stage I (limited invasion without regional lymph node metastasis), but decreases to below 20 % in the UICC stage IV (presence of distant metastasis).
  • Neoadjuvant and adjuvant chemotherapeutic and radiotherapeutic strategies are used to prevent locoregional and distant recurrences, but are effective only in a fraction of stage IV CRC patients.
  • Chemotherapy can lead to a partial remission of distant metastases and can enable secondary palliative surgeries and thereby result in long-term survival.
  • Approximately 25,000 metastatic colorectal cancer patients receive palliative chemotherapy in Germany every year.
  • Clinical decisions on the therapeutic procedure and extent of resectional treatment in colorectal carcinoma are presently based on imaging and on conventional histopathological features. The diagnostic accuracy of these approaches is limited, which leads to surgical interventions that are most often more radical than required, or to chemotherapeutic treatment of patients who do not benefit from this harsh regimen.
  • irinotecan a semisynthetic camptothecin, which inhibits topoisomerase I
  • oxaliplatin a third generation platinum compound
  • bevacizumab a monoclonal antibody directed against vascular endothelial growth factor (VEGF)
  • VEGF vascular endothelial growth factor
  • F-spondin (SPON-1, Spondin 1) has been identified as a protein expressed and secreted at high levels in the floor plate, a cell group implicated in the control of neural cell pattern and axonal growth in the developing vertebrate nervous system.
  • the bovine homolog of F-spondin has been identified independently as VSGP (Vascular smooth muscle cell growth-promoting factor).
  • M-spondin (SPON-2, Spondin 2, DIL-1, DIL1, Mindin) is an extracellular matrix protein highly homologous to F-spondin.
  • EGFR epidermal growth factor receptor
  • ErbB-1 is a cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands.
  • the epidermal growth factor receptor is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). It has been reported that mutations affecting EGFR expression or activity often result in cancer.
  • EGFR is a transmembrane protein receptor which is activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor ⁇ (TGF ⁇ ). Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer, which then stimulates cell growth, tissue proliferation and cell mitosis, the mechanism of which will be described in the following.
  • TGF ⁇ transforming growth factor ⁇
  • the said EGFR comprises a tyrsoine kinase on its intracellular domain.
  • EGFR dimerization stimulates the activity of said tyrosine kinase.
  • autophosphorylation of five tyrosine (Y) residues in the C-terminal domain of EGFR occurs. These are Y992, Y1045, Y1068, Y1148 and Y1173.
  • This autophosphorylation elicits downstream activation and signaling by several other proteins that associate with the phosphorylated tyrosines through their own phosphotyrosine-binding SH2 domains.
  • These downstream signaling proteins initiate several signal transduction cascades, principally the MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation (Shepherd, 2005) 5 .
  • VEGF Vascular Endothelial Growth Factor
  • EGFR may pair with another member of the ErbB receptor family, such as ErbB2/Her-2/neu, to create an activated heterodimer.
  • ErbB2/Her-2/neu another member of the ErbB receptor family
  • the overexpression of EGFR leads to an elevated abundance of said receptor in the cellular membranes, which leads to autonomous dimerization due to high receptor density, without the need for the ligand to elicit said dimerization.
  • an overexpression of either native or mutant EGFR due is frequently found in cancerogenic and pre-cancerogenic cells and/or tissues.
  • Said overexpression may be accompanied by mutations of the EGFR gene itself, as well as to gene amplification, polysomy, aneuploidy, genomic instability and the like.
  • Said overexpression leads to a self-activation of cell proliferation in the respective cells and/or tissues due to autonomous dimerization, as well as to the enhanced secretion of VEGF, which in turn stimulates cell proliferation in the very same cells and/or tissues, as well as to an enhanced vascularization of the respective tissue due to an enhanced angiogenesis.
  • Overexpression of EGFR does thus trigger a positive feedback mechanism which rapidly enforces tumor growth.
  • VEGFA overexpression VEGFA is frequently found in cancerogenic and pro-cancerogenic lesions, primary tumors and/or metatstatic lesions thereof. Said overexpression may be accompanied by enhanced receptor tyrosin kinase activities of tumor and/or adjacent stroma cells. VEGFA and it's family members may act as an autocrine or paracrine stimulus leading to enhanced cell proliferation, cell migration, repair capacity and or cell survival. Depending on the presence of receptors for said ligands VEGF factors may act on tumor cells, endothelial cells or other components of the stroma. Drugs inhibiting the VEGF ligand and VEGF receptor induced activities have been developed, such as small molecule inhibitors (e.g.
  • Sutent® and Nexaxar® or antibodies (e.g. Avastin®).
  • Avastin® e.g. Avastin®
  • EGFR family members similar to the situation described above for EGFR family members, their expression is difficult to reliably assess by standard technologies like immunohostochemistry and mRNA analysis methods like DNA microarrays or PCR methodologies. In part this is due to the limited dynamic range of the expression levels leading to different tumorbiological behaviour.
  • VEGFA is a member of the VEGF (Vascular endothelial growth factor) and related to the PDGF (Platelet derived growth factor) and FGF (Fibrobast growth factor) family, it can be assumed that the above mentioned mechanisms are also applicable to the other growth factors of said families.
  • VEGF Vascular endothelial growth factor
  • PDGF Plateelet derived growth factor
  • FGF Fibrobast growth factor
  • VEGFR1 is a member of the VEGFR (Vascular endothelial growth factor receptor) family, it can be assumed that the above mentioned mechanisms are also applicable to the other growth factors of said families, including the PDGFR and FGF Receptor family.
  • VEGFR Vascular endothelial growth factor receptor
  • prediction relates to an individual assessment of the malignancy of a tumor, or to the expected survival rate (DFS, disease free survival) of a patient, if the tumor is treated with a given therapy.
  • prognosis relates to an individual assessment of the malignancy of a tumor, or to the expected survival rate (DFS, disease free survival) of a patient, if the tumor remains untreated.
  • clinical response of a patient relates to the effectiveness of a certain therapy in a patient, meaning an improvement in any measure of patient status, including those measures ordinarily used in the art, such as overall survival, progression free survival, recurrence-free survival, and distant recurrence-free survival.
  • carcinomas e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma
  • pre-malignant conditions neomorphic changes independent of their histological origin (e.g. ductal, lobular, medullary, mixed origin).
  • carcinomas e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma
  • pre-malignant conditions neomorphic changes independent of their histological origin.
  • carcinomas e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma
  • pre-malignant conditions neomorphic changes independent of their histological origin.
  • cancer is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation. In particular stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer and primary carcinomas are included. Examples of cancers include, but are not limited to colorectal cancer, lung cancer, ovarian cancer, cervical cancer, stomach cancer, pancreatic cancer, head and neck cancer and/or breast cancer.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • determining the status refers to a measurable property of a gene and its products, especially on the nucleotide level and the gene level including mutation status and gene expression status.
  • a number of parameters to determine the status of a gene and its products can be used including, but not limited to, determining the level of protein expression, the amplification or expression status on RNA level or DNA level, of polynucleotides and of polypeptides, and the analysis of haplotype or the mutation status of the gene.
  • An exemplary determinable property correlated with the status of estrogen receptor or progesterone receptor is the amount of the estrogen receptor or progesterone receptor RNA, DNA or other polypeptide in the sample or the presence of nucleotide polymorphisms.
  • biological sample refers to a sample obtained from a patient.
  • the sample may be of any biological tissue or fluid.
  • samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue, core or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, urine, peritoneal fluid, and pleural fluid, or cells there from.
  • Biological samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof.
  • a biological sample to be analyzed is tissue material from neoplastic lesion taken by aspiration or punctuation, excision or by any other surgical method leading to biopsy or resected cellular material.
  • Such biological sample may comprise cells obtained from a patient.
  • the cells may be found in a cell "smear" collected, for example, by a nipple aspiration, ductal lavarge, fine needle biopsy or from provoked or spontaneous nipple discharge.
  • the sample is a body fluid.
  • Such fluids include, for example, blood fluids, serum, plasma, lymph, ascitic fluids, gynecological fluids, or urine but not limited to these fluids.
  • therapy modality refers to a timely sequential or simultaneous administration of anti-tumor, and/or immune stimulating, and/or blood cell proliferative agents, and/or radiation therapy, and/or hyperthermia, and/or hypothermia for cancer therapy.
  • the administration of these can be performed in an adjuvant and/or neoadjuvant mode.
  • the composition of such "protocol” may vary in the dose of the single agent, timeframe of application and frequency of administration within a defined therapy window.
  • various combinations of various drugs and/or physical methods, and various schedules are under investigation.
  • array or “matrix” is meant an arrangement of addressable locations or “addresses” on a device.
  • the locations can be arranged in two dimensional arrays, three dimensional arrays, or other matrix formats.
  • the number of locations can range from several to at least hundreds of thousands. Most importantly, each location represents a totally independent reaction site.
  • Arrays include but are not limited to nucleic acid arrays, protein arrays and antibody arrays.
  • a “nucleic acid array” refers to an array containing nucleic acid probes, such as oligonucleotides, polynucleotides or larger portions of genes.
  • the nucleic acid on the array is preferably single stranded.
  • oligonucleotide arrays wherein the probes are oligonucleotides are referred to as "oligonucleotide arrays" or “oligonucleotide chips.”
  • a “microarray,” herein also refers to a “biochip” or “biological chip”, an array of regions having a density of discrete regions of at least about 100/cm 2 , and preferably at least about 1000/cm 2 . The regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 ⁇ m, and are separated from other regions in the array by about the same distance.
  • a “protein array” refers to an array containing polypeptide probes or protein probes which can be in native form or denatured.
  • An “antibody array” refers to an array containing antibodies which include but are not limited to monoclonal antibodies (e.g. from a mouse), chimeric antibodies, humanized antibodies or phage antibodies and single chain antibodies as well as fragment
  • small molecule is meant to refer to a compound which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
  • Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules.
  • Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate a bioactivity.
  • regulated or “regulation” and “differentially regulated” as used herein refer to both upregulation [i.e., activation or stimulation (e.g., by agonizing or potentiating] and down regulation [i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)].
  • transcriptome relates to the set of all messenger RNA (mRNA) molecules, or "transcripts", produced in one or a population of cells.
  • the term can be applied to the total set of transcripts in a given organism, or to the specific subset of transcripts present in a particular cell type.
  • the transcriptome can vary with external environmental conditions. Because it includes all mRNA transcripts in the cell, the transcriptome reflects the genes that are being actively expressed at any given time, with the exception of mRNA degradation phenomena such as transcriptional attenuation.
  • the discipline of transcriptomics examines the expression level of mRNAs in a given cell population, often using high-throughput techniques based on DNA microarray technology.
  • expression levels refers, e.g., to a determined level of gene expression.
  • pattern of expression levels refers to a determined level of gene expression compared either to a reference gene (e.g. housekeeper or inversely regulated genes) or to a computed average expression value (e.g. in DNA-chip analyses).
  • a pattern is not limited to the comparison of two genes but is more related to multiple comparisons of genes to reference genes or samples.
  • a certain "pattern of expression levels” may also result and be determined by comparison and measurement of several genes disclosed hereafter and display the relative abundance of these transcripts to each other.
  • a differentially expressed gene disclosed herein may be used in methods for identifying reagents and compounds and uses of these reagents and compounds for the treatment of cancer as well as methods of treatment.
  • the differential regulation of the gene is not limited to a specific cancer cell type or clone, but rather displays the interplay of cancer cells, muscle cells, stromal cells, connective tissue cells, other epithelial cells, endothelial cells of blood vessels as well as cells of the immune system (e.g. lymphocytes, macrophages, killer cells).
  • a "reference pattern of expression levels”, within the meaning of the invention shall be understood as being any pattern of expression levels that can be used for the comparison to another pattern of expression levels.
  • a reference pattern of expression levels is, e.g., an average pattern of expression levels observed in a group of healthy or diseased individuals, serving as a reference group.
  • Primer pairs and “probes”, within the meaning of the invention, shall have the ordinary meaning of this term which is well known to the person skilled in the art of molecular biology.
  • “primer pairs” and “probes” shall be understood as being polynucleotide molecules having a sequence identical, complementary, homologous, or homologous to the complement of regions of a target polynucleotide which is to be detected or quantified.
  • nucleotide analogues are also comprised for usage as primers and/or probes.
  • arrayed probes within the meaning of the invention, shall be understood as being a collection of immobilized probes, preferably in an orderly arrangement.
  • the individual “arrayed probes” can be identified by their respective position on the solid support, e.g., on a "chip”.
  • therapeutic success refers, in the adjuvant chemotherapeutic setting to the observation of a defined tumor free or recurrence free survival time (e.g. 2 years, 4 years, 5 years, 10 years). This time period of disease free survival may vary among the different tumor entities but is sufficiently longer than the average time period in which most of the recurrences appear.
  • response may be monitored by measurement of tumor shrinkage due to apoptosis and necrosis of the tumor mass.
  • recurrence includes distant metastasis that can appear even many years after the initial diagnosis and therapy of a tumor, or local events such as infiltration of tumor cells into regional lymph nodes, or occurrence of tumor cells at the same site and organ of origin within an appropriate time.
  • Prediction of therapeutic success does refer to the methods described in this invention. Wherein a tumor specimen is analyzed for it's gene expression and furthermore classified based on correlation of the expression pattern to known ones from reference samples. This classification may either result in the statement that such given tumor will develop recurrence and therefore is considered as a "non responding" tumor to the given therapy, or may result in a classification as a tumor with a prolonged disease free post therapy time.
  • marker refers to a biological molecule, e.g., a nucleic acid, peptide, protein, hormone, etc., whose presence or concentration can be detected and correlated with a known condition, such as a disease state.
  • ligand relates to a molecule that is able to bind to and form a complex with a biomolecule to serve a biological purpose. In a narrower sense, it is an effector molecule binding to a site on a target protein, by intermolecular forces such as ionic bonds, hydrogen bonds and Van der Waals forces.
  • the docking (association) is usually reversible (dissociation).
  • Actual irreversible covalent binding between a ligand and its target molecule is rare in biological systems.
  • Ligand binding to receptors often alters the chemical conformation, i.e. the three dimensional shape of the receptor protein. The conformational state of a receptor protein determines the functional state of a receptor. The tendency or strength of binding is called affinity.
  • Ligands include substrates, inhibitors, activators, and neurotransmitters.
  • agonist relates to a substance that binds to a specific receptor and triggers a response in the cell. It mimics the action of an endogenous ligand that binds to the same receptor.
  • receptor relates to a protein on the cell membrane or within the cytoplasm or cell nucleus that binds to a specific molecule (a ligand), such as a neurotransmitter, hormone, or other substance, and initiates the cellular response to the ligand.
  • a ligand such as a neurotransmitter, hormone, or other substance
  • signalling pathway is related to any intra- or intercellular process by which cells converts one kind of signal or stimulus into another, most often involving ordered sequences of biochemical reactions out- and inside the cell, that are carried out by enzymes and linked through hormones and growth factors (intercellular), as well as second messengers (intracellular), the latter resulting in what is thought of as a "second messenger pathway".
  • intercellular hormones and growth factors
  • intracellular second messengers
  • the number of proteins and other molecules participating in these events increases as the process emanates from the initial stimulus, resulting in a "signal cascade” and often results in a relatively small stimulus eliciting a large response.
  • marker gene refers to a differentially expressed gene whose expression pattern may be utilized as part of a predictive, prognostic or diagnostic process in malignant neoplasia or cancer evaluation, or which, alternatively, may be used in methods for identifying compounds useful for the treatment or prevention of malignant neoplasia and colorectal cancer, lung cancer, ovarian cancer, cervical cancer, stomach cancer, pancreatic cancer, head and neck cancer and/or breast cancer in particular.
  • a marker gene may also have the characteristics of a target gene.
  • Target gene refers to a differentially expressed gene involved in cancer, preferably colorectal cancer, in a manner in which modulation of the level of the target gene expression or of the target gene product activity may act to ameliorate symptoms of cancer, preferably colorectal cancer.
  • a target gene may also have the characteristics of a marker gene.
  • expression level relates to the process by which a gene's DNA sequence is converted into functional protein and particularly to the amount of said conversion.
  • substantially homologous refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • hybridization based method refers to methods imparting a process of combining complementary, single-stranded nucleic acids or nucleotide analogues into a single double stranded molecule. Nucleotides or nucleotide analogues will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily. In bioanalytics, very often labeled, single stranded probes are in order to find complementary target sequences. If such sequences exist in the sample, the probes will hybridize to said sequences which can then be detected due to the label. Other hybridization based methods comprise microarray and/or biochip methods. Therein, probes are immobilized on a solid phase, which is then exposed to a sample. If complementary nucleic acids exist in the sample, these will hybridize to the probes and can thus be detected.
  • hybridization based methods are also known as “array based methods”.
  • PCR is another hybridization based method.
  • hybridization based methods may for example be used to determine the amount of mRNA for a given gene.
  • a PCR based method refers to methods comprising a polymerase chain reaction (PCR). This is an approach for exponentially amplifying nucleic acids, like DNA or RNA, via enzymatic replication, without using a living organism. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations. When it comes to the determination of expression levels, a PCR based method may for example be used to detect the presence of a given mRNA by (1) reverse transcription of the complete mRNA pool (the so called transcriptome) into cDNA with help of a reverse transcriptase enzyme, and (2) detecting the presence of a given cDNA with help of respective primers. This approach is commonly known as reverse transcriptase PCR (rtPCR).
  • rtPCR reverse transcriptase PCR
  • determining the protein level refers to methods which allow the quantitative and/or qualitative determination of one or more proteins in a sample. These methods include, among others, protein purification, including ultracentrifugation, precipitation and chromatography, as well as protein analysis and determination, including the use protein microarrays, two-hybrid screening, blotting methods including western blot, mass spectrometry, one- and two dimensional gelelectrophoresis, isoelectric focusing and the like.
  • anamnesis relates to patient data gained by a physician or other healthcare professional by asking specific questions, either of the patient or of other people who know the person and can give suitable information (in this case, it is sometimes called heteroanamnesis), with the aim of obtaining information useful in formulating a diagnosis and providing medical care to the patient. This kind of information is called the symptoms, in contrast with clinical signs, which are ascertained by direct examination.
  • the term "etiopathology” relates to the course of a disease, that is its duration, its clinical symptoms, and its outcome.
  • this refers to therapies targeting ERB receptor family members itself.
  • this refers to gene products regulated by ERB family members and downstream activities thereof, such as family members of the VEGF/VEGFR and/or FGF/FGFR system.
  • a method for predicting a clinical response of a patient suffering from or at risk of developing cancer, preferably colorectal cancer, towards a given mode of treatment comprising the steps of:
  • said cancer is selected from the group comprising colorectal cancer, lung cancer, ovarian cancer, cervical cancer, stomach cancer, pancreatic cancer, head and neck cancer and/or breast cancer.
  • said cancer is colorectal cancer.
  • said colorectal cancer is a stage III or stage IV colorectal cancer, in particular a primary tumor.
  • upregulated expression of SPON-1 and/or SPON-2 is indicative of a poor prediction as regards therapeutic success for patients receiving a chemotherapy.
  • the inventors of the present invention found that there is a trend in a yet small stage III tumor cohort indicating SPON-1 to predict early recurrence despite adjuvant chemotherapy treatment.
  • SPON-1 and SPON-2 provide an improved method to predict response to chemotherapy on expression profiling.
  • stage III tumors are superior to the results obtained by using SPON-1.
  • upregulated expression of SPON-1 and/or SPON-2 is indicative of a promising prediction as regards therapeutic success for patients receiving a therapeutic regimen targeting the signalling pathway of receptors from the VEGF receptor and/or ligand family.
  • endothelial cell and smooth muscle cell growth factors are highly expressed in areas undergoing significant angiogenesis. Since angiogenesis is also required to support tumor growth, the inventors of the present invention suggest to determine the expression level of at least SPON-2, optionally combined with the expression level of SPON-1, in a sample to predict response to therapeutic regimen targeting the signalling pathway of receptors from the VEGF receptor and/or ligand family.
  • upregulated expression of SPON-1 and/or SPON-2 is indicative of a promising prediction as regards therapeutic success for patients receiving a therapeutic regimen targeting the signalling pathway of receptors from the ErbB receptor and/or ligand family.
  • the mode of treatment for which prediction is sought is a treatment related to the signalling pathway of receptors from the ErbB receptor and/or ligand family.
  • the ErbB family of receptors comprises four closely related receptor tyrosine kinases, namely
  • a preferred receptor of the method according to the invention is EGFR, as there is
  • the PDGF family of growth factors comprises several members which all have in common that they feature a cystine-knot domain, and bind to tyrosine kinase receptors, like those from the ErbB family.
  • the VEGF family comprises members of the VEGF sub-family, i.e.
  • VEGF-E viruses
  • VEGF-F venom of some snakes
  • All of these growth factors are ligands which are related to the ErbB signalling pathway, as their expression level is upregulated upon activation or self activation of a receptor of the ErbB family, particularly of EGFR.
  • the growths factors do thus meet the above identified definition according to which the said ligand is related to the signalling pathway of receptors from the ErbB receptor family.
  • the mode of treatment for which prediction is sought is a treatment related to the signalling pathway of receptors from the VEGF receptor and/or ligand family.
  • said given mode of treatment (a) acts on recruitment of lymphatic vessels, cell proliferation, cell survival, cell repair mechanisms and/or cell motility, and/or b) comprises administration of a chemotherapeutic agent.
  • said given mode of treatment comprises chemotherapy, in particular palliative chemotherapy, administration of small molecule inhibitors, antibody based regimen, anti-proliferation regimen, proapoptotic regimen, pro-differentiation regimen, radiation and/or surgical therapy.
  • method of selecting a therapy modality for a patient afflicted with cancer preferably colorectal cancer, comprising the steps of:
  • determining the expression level comprises:
  • the said expression level is determined by
  • the expression level of at least one of the said marker genes is determined with rtPCR (reverse transcriptase polymerase chain reaction) of the related mRNA.
  • the mRNA related to the markers is determined, namely with a reverse transcriptase polymerase chain reaction approach.
  • the inventors of the present invention have surprisingly found out that the determination of the marker genes' mRNA levels is very informative for the prediction of the clinical response of a patient suffering from or at risk of developing cancer, preferably colorectal cancer, towards a medicament.
  • anti-ErbB/anti-EGFR treatments for example the anti-VEGF antibody Bevacizumab
  • anti-PDGF/anti-VEGF treatments for example the anti-VEGF antibody Bevacizumab
  • VEGF-related processes may be considered as one symptom out of a range of several symptoms related to tumor genesis, whereas defects in EGFR expression may be considered as the underlying cause for these symptoms, the treatment of which seems thus to be much more promising than the treatment of just one of said symptoms.
  • the expression level of at least one of the said marker genes is determined in formalin and/or paraffin fixed tissue samples.
  • the expression level of at least one of the said ligands or receptors is determined in serum, plasma or whole blood samples.
  • RNA samples are taken as biopsies form a patient and undergo diagnostic procedures.
  • the samples are fixed in formalin and/or paraffin and are then examined with immunohistochemistry methods.
  • the formalin treatment leads to the inactivation of enzymes, as for example the ubiquitous RNA-digesting enzymes (RNAses).
  • RNAses ubiquitous RNA-digesting enzymes
  • the mRNA status of the tissue remains undigested.
  • the samples are treated with silica-coated magnetic particles and a chaotropic salt, in order to purify the nucleic acids contained in said sample for further determination.
  • Collaborators of the inventors of the present invention have developed an approach which however allows successful purification of mRNA out of tissue samples fixed in such manner, and which is disclosed, among others, in WO03058649 , WO2006136314A1 and DE10201084A1 , the content of which is incorporated herein by reference.
  • Said method comprises the use of magnetic particles coated with silica (SiO 2 ).
  • the silica layer is closed and tight and is characterized by having an extremely small thickness on the scale of a few nanometers.
  • These particles are produced by an improved method that leads to a product having a closed silica layer and thus entail a highly improved purity.
  • the said method prevents an uncontrolled formation of aggregates and clusters of silicates on the magnetite surface whereby positively influencing the additional cited properties and biological applications.
  • the said magnetic particles exhibit an optimized magnetization and suspension behavior as well as a very advantageous run-off behavior from plastic surfaces.
  • These highly pure magnetic particles coated with silicon dioxide are used for isolating nucleic acids, including DNA and RNA, from cell and tissue samples, the separating out from a sample matrix ensuing by means of magnetic fields. These particles are particularly well-suited for the automatic purification of nucleic acids, mostly from biological body samples for the purpose of detecting them with different amplification methods.
  • the said approach is particularly useful for the purification of mRNA out of formalin and/or paraffin fixed tissue samples.
  • the said approach creates mRNA fragments which are large enough to allow specific primer hybridization and/or specific probe hybridization.
  • a minimal size of at least 100 bp, more preferably 200 base pairs is needed for specific and robust detection of target gene expression.
  • Other issues of perturbance of expression data by sample preparation problems relate to the contamination level with DNA, which is lower compared to other bead based technologies. This of particular importance, as the inventors have observed, that DNAse treatment is not efficient in approximately 10% of FFPE samples generated by standard procedures and stored at room temperature for some years before cutting and RNA extraction.
  • the said approach thus allows a highly specific determination of candidate gene expression levels with one of the above introduced methods, particularly with hybridization based methods, PCR based methods and/or array based methods, even in formalin and/or paraffin fixed tissue samples, and is thus extremely beneficial in the context of the present invention, as it allows the use of tissue samples fixed with formalin and/or paraffin, which are available in tissue banks and connected to clinical databases of sufficient follow-up to allow retrospective analysis.
  • said treatment related to the signalling pathway of receptors from the ErbB receptor family comprises the administration of an agonist of the ErbB receptor domain.
  • said treatment related to the signalling pathway of receptors from the ErbB receptor family comprises the administration of nucleotides, ribozomes, aptamers and /or nucleotide analogues capable of affecting the expression of ErbB receptor, VEGF receptor and /or VEGFA ligand expression.
  • these substances act via downregulation of mRNA levels of said candidate genes.
  • induction or elevation of gene expression by introduction of receptor gene expression can be advantageous to counteract the tumorpromoting activity (e.g. of EGFR, Her-2/neu and/or VEGFC).
  • said agonists may be selected from the group consisting of Cetuximab (tradename Erbitux®, target receptor is EGFR), Matuzumab (EMD7200, target receptor is EGFR), Trastuzumab (tradename Herceptin®, target receptor is HER2/neu), Pertuzumab (target receptor is HER2/neu), Bevacizumab (tradename Avastin®, target ligand is VEGFA), 2C3 (target ligand is VEGFA), VEGF-trap (AVE-0005, target ligands are VEGFA and PIGF), IMC-1121B (target receptor is VEGFR2), and CDP-791 (target receptor is VEGFR2).
  • said treatment related to the signalling pathway of receptors from the ErbB receptor family comprises the administration of a tyrosin kinase inhibitor.
  • said treatment related to the signalling pathway of receptors from the VEGF receptor family comprises the administration of an agonist of the VEGF receptor domain.
  • said inhibitors may be selected from the group consisting of Gefitinib (tradename Iressa®, ZD-1839, target receptor is EGFR), Erlotinib (tradename Tarceva®, OSI-774, target receptor is EGFR), EKB-569 (target receptor is EGFR), PKI-166 (target receptor is EGFR), ), PKI-166 (target receptor is EGFR), Lapatinib (tradename tycerb®, target receptor is EGFR and Her-2/neu), GW572016 (target receptors are EGFR and Her-2/neu), AEE-788 (target receptors are EGFR, Her-2/neu and VEGFR-2), CI-1033 (target receptors are EGFR, Her-2/neu and Her4), and AZD6474 (target receptors are EGFR and VEGFR-2).
  • Target receptors are VEGFR-2, VEGFR-3, c-KIT, PDGFR-B, RET and Raf-Kinase
  • BAY 57-9352 target receptor is VEGFR-2
  • Sunitinib tradename Sutent®, target receptors are VEGFR-1, VEGFR-2 and PDGFR
  • AG13925 target receptors are VEGFR-1 and VEGFR-2
  • AG013736 target receptors are VEGFR-1 and VEGFR-2
  • AZD2171 target receptors are VEGFR-1 and VEGFR-2
  • ZD6474 target receptors are VEGFR-1, VEGFR-2 and VEGFR-3
  • PTK-787/ZK-222584 target receptors are VEGFR-1 and VEGFR-2
  • CEP-7055 target receptors are VEGFR-1, VEGFR-1, VEGFR-1, VEGFR-3
  • the treatment comprises the administration of chemotherapeutics.
  • Said chemotherapeutics may be selected from the group consisting of Cyclophosphamid (Endoxan®, Cyclostin®).
  • Adriamycin Doxorubicin
  • BCNU Carmustin
  • Busulfan Myleran®
  • Bleomycin Bleomycin
  • Carboplat® Chlorambucil (Leukeran®)
  • CisPlatin CisPlatin
  • Platinex Platinum
  • Tabletiblastin® dacarbazin
  • Docetaxel Taxotere®
  • Epirubicin Flumorubicin®
  • Etoposid Etoposid
  • Vepesid® 5-Fluorouracil
  • Fluorouracil® Fluorouracil®
  • Gemcitabin Gamzar®
  • kit useful for carrying out one of the said methods comprising at least
  • a method for correlating the clinical outcome of a patient suffering from or at risk of developing cancer, preferably colorectal cancer, with the presence or non-presence of a defect in expression of at least one gene selected from the group consisting of SPON-1 and/or SPON-2 comprising the steps of:
  • the said method is particularly beneficial for epidemiological studies. These studies profit from the fact that large tissue databases exist comprising paraffin and/or formalin fixed tissue samples together with an extensive documentation of the patient's history, including etiopathology data, clinical symptoms, anamnesis data and/or data concerning the therapeutic regimen.
  • the said methods allow for large scale studies which comprise the correlation of the clinical outcome of a patient suffering from or at risk of developing cancer, preferably colorectal cancer, with the presence or non-presence of a defect in expression of at least one of the marker genes according to the present invention.
  • the above introduced method for mRNA purification comprising silica coated magnetic beads and chaotropic salts is quite helpful.
  • nucleic acid primer pair and/or a nucleic acid probe having a sequence sufficiently complementary to a gene encoding for SPON-1 and/or SPON-2 is disclosed.
  • an antibody directed against SPON-1 and/or SPON-2 is disclosed.
  • Intraoperatively obtained biopsies from colorectal primary tumors and liver metastases were shock-frozen with liquid nitrogen immediately (within one minute after removal) and then stored at - 80°C.
  • the frozen tissues were cut into 8 m sections using a cryostat and then stained with hematoxylin and eosin for histological examination.
  • Laser capture microdissection (LCM) was performed immediately after staining and dehydration. Tumor areas of interest were selected with the help of an experienced pathologist (A.D.) and excised using a 0.6 mm laser beam (32 mW, 30 Hz, 0.8 sec pulse). Each sample yielded approximately 10.000 cells.
  • Captured cells were dissolved in RLT buffer (RNeasy Mini Kit, Qiagen, Hilden, Germany) and RNA was extracted as described below. Tumor material from two colon primary tumors and one liver metastasis were differentially microdissected into stromal cells and tumor cells.
  • each biopsy yielded up to 800 ng of total RNA.
  • each sample typically provided a final yield of 50-100 g of amplified RNA (aRNA).
  • aRNA amplified RNA
  • Samples were hybridised to Affymetrix HG U133-A high-density oligonucleotide-based arrays (Affymetrix, Santa Clara/CA, USA) targeting 22,230 human genes and expressed sequence tags (EST). From each biopsy, 15 g of either cRNA or aRNA was loaded onto an array following the recommended procedures for prehybridization, hybridization, washing and staining with streptavidin-phycoerythrin. The arrays were scanned on an Affymetrix GeneChip Scanner (Agilent, Palo Alto, CA). The fluorescence intensity was measured for each microarray and normalised to the average fluorescence intensity of the entire microarray.
  • the raw, unnormalized data-sets were analyzed by MicroArray Suite (Affymetrix) for normalization and estimation of expression values.
  • Signal intensities and detection calls for statistical analysis and hierarchical clustering were determined using the GeneChip 5.0 software (Affymetrix) and Expressionist TM software (Genedata).
  • Unsupervised two-dimensional hierarchical cluster analysis of the candidate gene data were done using the between-groups linkage method with the 2 measure for ordinal data to identify individual groups of tumours with specific SPON-1 and/or SPON-2 profiles.
  • Significance levels of microarray results for responding vs. non-responding Stage IV colorectal cancer or recurrent vs. non-recurrent Stage III colorectal cancer were calculated using the Mann-Whitney U-test. A p value of ⁇ 0.05 was regarded as significant.
  • the FFPE slide is deparraffinized in xylol and ethanol, the pellet is washed with ethanol and dried at 55°C for 10 minutes. The pellet is then lysed and proteinized overnight at 55°C with shaking. After adding a binding buffer and the magnetic particles (Siemens Medical Solutions Diagnostic GmbH, Leverkusen, Germany) nucleic acids are bound to the particles within 15 minutes at room temperature.
  • RT-PCR reverse transcription-polymerase chain reaction
  • RNA-specific Primer/Probe were designed to detect all isoforms simultaneously. Expression of each gene was defined as high and low according to values above and up to the median, respectively.
  • SPON-2 is a more appropriate marker to discriminate recurrent versus non-recurrent stage III colorectal tumors.
  • Said statement can be further strengthened by comparison of the expression data of SPON-1 and SPON-2 in stage III colorectal cancer patients ( Figures 3 and 4 , patients with a recurrent disease are marked with an asterisk*, and patients with a non-recurrent disease are not marked with an asterisk*).
  • SPON-2 as gene marker patients with a recurrent diesease could be identified improved compared to SPON-1.
  • SPON-2 By expression analysis of SPON-2, optionally combined with SPON-1, an improved molecular test to select a more appropriate therapy can be provided.
  • SPON-1 has been identified to be upregulated in ovarian cancer (Pyle-Chenault et al., 2005) 8 .
  • VEGF appears to play a major role in the formation of new vessels by stimulating endothelial cell proliferation and migration (Hazzard and Stouffer, 2000) 9 .
  • the identification of SPON-1 in normal bovine ovarian follicular fluid (Miyamoto et al., 2001) 10 indicates that SPON-1 may complement VEGF activity during angiogenesis. Pyle-Chenault et al. concluded that endothelial cell and smooth muscle cell growth factors would be highly expressed in areas undergoing significant angiogenesis.
  • the inventors of the present invention have for the first time suggested not to use the targets themselves as marker genes, but SPON-2, optionally combined with SPON-1, as marker genes to predict the clinical response of a patient suffering from or at risk of developing colorectal cancer towards a medicament related to the signalling pathway of receptors from the ErbB receptor family.
  • Table 1 Primer and Probe Sequences of Interest Internal number Gene NM_number Probe Forward primer Reverse primer SC045 (SEQ ID NOs: 1-3) SPON-1 NM_00610 8 SC297 (SEQ ID NOs: 4-6) SPON-1 NM_00610 8 SC298 (SEQ ID NOs: 7-9) SPON-1 NM_00610 9 SC299 (SEQ ID NOs: 10-12) SPON-2 NM_01244 5 SC300 (SEQ ID NOs: 13-15) SPON-2 NM_01244 6
  • Table 2 Spearman Rho correlation between SPON-1 and SPON-2 Variable 1 Variable 2 Spearman Rho SPON-1 SPON-1 SC_45_manual 0.7335 SC_297_manual SPON-2 SPON-1 SC_45_manual 0.5447 SC_299_manual SPON-2 SPON-1 0.1677 SC_299_manual SC_297_manual SPON-2 SPON-1 SC_45_manual 0.5940 SC_300_manual SP

Claims (6)

  1. Procédé de prédiction d'une réaction clinique d'un patient souffrant d'un cancer colorectal vis-à-vis d'un mode donné de traitement, le procédé comprenant les stades :
    a ) de détermination du taux d'expression d'au moins SPON-2, et facultativement de détermination du taux d'expression de SPON-1, dans un échantillon biologique d'un patient ;
    b) de comparaison du taux d'expression ou des taux d'expression déterminé ( s ) dans a ) à un ou à plusieurs taux d'expression témoin ( s ) et
    c ) de prédiction du succès thérapeutique du mode donné de traitement chez le patient ou de mise en oeuvre d'un régime thérapeutique chez le sujet, à partir du résultat de la comparaison dans le stade b ), caractérisé en ce que le cancer colorectal est un cancer colorectal du stade III, en particulier une tumeur primaire, et en ce que l'expression sur-régulée d'au moins SPON-2, et facultativement de SPON-1, est indicatrice d'une prédiction médiocre en ce qui concerne le succès thérapeutique pour des patients recevant une chimiothérapie.
  2. Procédé suivant la revendication 1, caractérisé en ce qu'une expression sur-régulée d'au moins SPON-2, et facultativement de SPON-1, est indicatrice d'une rechute de tumeur colorectale du stade III chez des patients cancéreux recevant une chimiothérapie.
  3. Procédé suivant l'une quelconque des revendications précédentes, dans lequel déterminer le taux d'expression comprend :
    a ) déterminer le taux d'expression d'ANR et/ou
    b ) déterminer le taux d'expression de protéine.
  4. Procédé suivant l'une quelconque des revendications précédentes, dans lequel on détermine le taux d'expression par
    a ) un procédé reposant sur une hybridation,
    b ) un procédé reposant sur une PCR,
    c ) on détermine le taux de protéine et/ou par
    d ) un procédé reposant sur une répartition.
  5. Procédé suivant l'une quelconque des revendications précédentes, caractérisé en ce que l'on détermine le taux d'expression d'au moins SPON-2, et facultativement de SPON-1, par PCRrt ( réaction en chaîne polymérase de transcriptase inverse ) de l'ARNm apparenté.
  6. Procédé de corrélation de l'évolution clinique d'un patient souffrant de cancer colorectal en la présence ou en la non présence d'un défaut d'expression d'au moins SPON-2, et facultativement de SPON-1, le procédé comprenant les stades :
    a ) de détermination du taux d'expression d'au moins SPON-2, et facultativement de SPON-1, dans un échantillon biologique ( fixé ) d'un patient suivant l'un quelconque des procédés ci-dessus et
    b ) de corrélation de l'évolution du ( des ) taux d'expression déterminé ( s ) dans a ) avec des données du patient, ces données étant choisies dans le groupe consistant en des données d'étiopathologie, des symptômes cliniques, des données d'anamnèse et/ou des données concernant le régime thérapeutique.
EP09737953.1A 2008-04-29 2009-03-27 Procédé de prédiction d'une réponse clinique d'un patient souffrant d'un cancer ou présentant un risque de développer un cancer vis-à-vis d'un mode de traitement donné Active EP2281063B1 (fr)

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